Platelet-derived growth factor (PDGF) is a major serum mitogen for cells of mesenchymal origin such as glial cells, fibroblasts, and smooth muscle cells (Heldin, et al., Cell Reg. 1: 555-566 (1990); Aaronson, S., Science 254; 1146-1153 (1991)). Biochemical evidence has established the existence in vivo of three PDGF isoforms; PDGF AA, PDGF BB, and PDGF AB (Johnson et al., Biochem. Biophys. Res. Commun. 104: 66-71 (1982)). PDGF BB is the human homologue of the v-sis oncogene product (Doolittle et al., Science 221: 275-277 (1983); Waterfield et al. Nature 304: 35-39 (1983); and Devare et al., Proc. Nat'l Acad. Sci. USA 80: 731-735 (1983)). Cell responsiveness to PDGF depends on signal transduction through cell surface .alpha. or .beta. PDGF receptors ("PDGFR's") (Matsui et al. Science 243: 800-804 (1989); Claesson-Welsh, et al., Proc. Nat'l Acad. Sci. USA 86: 4917-4921 (1989); and Yarden et al., Nature 323: 226-232 (1986)). The .alpha. PDGFR is triggered by all PDGF isoforms, while the .beta. PDGFR is activated by PDGF BB and to a lesser extent by PDGF AB (Matsui, et al. Proc. Nat'l Acad. Sci. USA 86: 8314-8318 (1989); Hart, et al. Science 240: 1529-1531 (1988); and Heldin, et al. EMBO J. 7: 1387-1393 (1988)).
In a previous study, expression vectors for the .alpha. and .beta. PDGFR cDNAs were introduced into an IL-3 dependent murine hematopoietic cell line, 32D, which did not normally express these genes (Matsui, et al. supra (1989)). Data revealed that each receptor independently coupled with signal transduction pathways inherently present in these cells. Furthermore, both receptors induced a readily detectable mitogenic and chemotactic responses. Activation of either PDGFR stimulated inositol phospholipid metabolism and mobilization of Ca++.
Although PDGF's physiological role is associated with wound healing and development, the interactions of PDGF with its cognate PDGFR have been implicated in neoplasia (Nister et al. J. Biol. Chem. 266:16755-16763 (1991) and Fleming et al., Oncogene 7:1355-1359 (1992)), arteriosclerosis (Ross et al., Science 248: 1009-1012 (1990) and Ferns et al., Science 253:1129-1132 (1991)). PDGF has also been identified in fibrotic diseases (Smits et al., Am. J. Path. 140: 639-648 (1992)) such as scleroderma (Gay et al., J. Invest. Dermatol. 92:301-303 (1989)) and glomerulonephritis (Iida et al., Proc. Natl. Acad. Sci. USA 88:6560-6564 (1991)).
Methods of isolating PDGF have involved the purification of PDGF from platelets as described in Johnsson et al., Biochem. Biophys. Res. Comm. 104: 66-74 (1982) and the development of antibodies against PDGF which are capable of detecting PDGF. For instance, Kelly et al. EMBO J. 4: 3399-3405 (1985) describe the production of polyclonal antibodies against PDGF but which are not capable of distinguishing the various isoforms of PDGF. Niman et al., Nature 307: 180-183 (1984) describe monoclonal antibodies raised against synthetic peptides derived from the amino terminal of the B-chain of PDGF which are not suitable for identifying intact, active forms of PDGF. U.S. Pat. No. 5,094,941 describe the production of monoclonal antibodies which recognize native PDGF and distinguish the three isoforms of PDGF. Vassbotn et al., Biochim. Biophy. Acta, 1054: 246-249 (1990) disclosed the production of monoclonal antibody mab 6D11 against PDGF which bound PDGF B--B and PDGF A-B and blocked binding with PDGFR. La Rochelle et al., Mol. Cell. Bio., 9:3538-3542 (1989) discloses the production of monoclonal antibody, sis 1, which recognizes human PDGF-BB and human PDGF-AB and inhibits PDGF receptor binding and mitogenic activities of PDGF-BB and PDGF-AB.
Similarly, poly- and monoclonal antibodies have been used to detect PDGFR's. For instance, Chaudhry et al., Cancer Research 52:1006-1012 (1992) discussed the use of polyclonal antibodies against the .alpha. PDGFR to study neuroendocrine tumors. Hart et al., J. Biol. Chem. 262: 10780-10785 (1987) and Seifert et al., J. Biol. Chem. 264: 8771-8778 (1989) both describe the production of monoclonal antibody PR7212 which binds to the .beta. PDGFR. Kawahara et al., Biochem. Biophys. Res. Comm. 147: 839-845 (1987) discloses the production of monoclonal antibody C3.1, raised against NR6 cells, which contain about 200,000 to 400,000 PDGFR's per cell. Kawahara showed that C3.1 acts as an antagonist by blocking PDGF stimulated mitogenesis, but they did not indicate where C3.1 binds the PDGFR. Ronnstrand et al., J. Biol. Chem. 263: 10429-10435 (1988) describe two monoclonal antibodies, denoted PDGFR-B1 and PDGFR-B2, and FAB' fragments thereof, that react with the PDGF receptor. Both antibodies recognized the extracellular part of the receptor, but neither blocked the binding of .sup.125 I-PDGF. A commercially available monoclonal antibody against .alpha. PDGFR is PR292, produced by Genzyme Corp., Cambridge, Mass. (product no. 1264-00). Although this monoclonal antibody is specific for the .alpha. PDGFR, it does not inhibit PDGF binding with its receptor.
Thus, a need exists for a potent neutralizing monoclonal antibody directed toward .alpha. PDGFR. Such a monoclonal antibody would be a potent antagonist capable of controlling or interfering with PDGF dependent autocrine growth associated with neoplasias, arteriosclerosis, fibrotic diseases and other pathological diseases known to be associated with .alpha. PDGFR expression.